Semen analysis

ABSTRACT

Provided is a method for measuring the total sperm concentration (TSC) in a sample including: (i) placing the sample in a transparent container between a synchronically pulsed light source and a photodetector; and (ii) measuring the optical absorbance of the sample in the range of 800-1000 nm, the TSC of the sample being proportional to the absorbance. Further provided is a sampling device for use in optically analyzing a biological fluid, a method for measuring motile sperm concentration (MSC) in a semen sample, a method of determining the average velocity (AV) of sperm cells and a system for analyzing semen quality comprising means for measuring TSC, means for measuring MSC; and a video visualization system.

FIELD OF THE INVENTION

This invention relates to semen analysis.

BACKGROUND OF THE INVENTION

According to WHO statistics, 8-10% of all married couples consultmedical professionals after failing to conceive. Over 40 million couplesare currently being treated for infertility. Among these infertilecouples, it is estimated that the infertility in 40% of the couples isdue to male originating causes, and another 20% is due to combined maleand female originating causes. Semen analysis is a major technique inevaluating male originating causes.

Standard semen analysis protocol involves the determination of at leastthree major semen parameters:

1. total sperm concentration (TSC);

2. percentage of motile sperm; and

3. percentage of normal sperm morphologies.

For all practical purposes, semen analysis, a key factor in human malefertility medicine, has not changed since the 1930's and is still donetoday by microscopic inspection. In fact, it is one of the very fewremaining in vitro, body fluid analysis still performed almost solelyvia manual methods.

This manual methodology involves carefully observing the sperm cells,counting them to determine their concentration, classifying theirmotility, identifying their morphology, etc. This work requires highexpertise, is very labor intensive and if done according to standardprotocols, takes at least an hour per test.

Manual assessments are known to be quite inaccurate due to numeroussources of error. The main sources of error are:

Subjectivity of the observer.

The varying criteria used in the different labs and by differentobservers.

The large statistical errors due to the limited number of spermanalyzed.

The WHO manual (WHO laboratory manual for the examination of human semenand sperm-cervical mucus interaction. 4^(th) edition, CambridgeUniversity Press, 1999) recommends observing not less than 200 sperm andclassifying the morphology and motility of each. This itself is an errorintroducing procedure due to the tediousness and time consuming natureof the task. In practice, 50 to 100 sperm cells at most are analyzed.Even if the observer introduces no errors, the statistical error alonereaches tens of percentages.

As a result of the above methodology, semen analysis test results areglobally recognized to be highly subjective, inaccurate and poorlyreproducible. Inter lab and inter technician variations are of suchproportions that this issue is of major concern in male fertilitymedicine and the unresolved subject of discussion in the vast majorityof symposiums, congresses and conventions on the subject.

In order to overcome these difficulties, medical instrumentationcompanies have introduced dedicated computerized systems based on imageanalysis (CASA—Computer Assisted Semen Analyzers). These systems requirean extremely high quality image because all their results are based onimage processing. Although these systems have attempted to replacemanual analysis and establish industry accepted standards, they have notsucceeded in either of these objectives.

The first objective could not be achieved because analysis resultscontinue to be dependent on manual settings and on the different makesof equipment. Replacing routine manual analysis is totally impracticablebecause the systems are extremely expensive, complex and difficult touse. The fact is that such systems are generally not found in routinesemen analysis but have rather established their niche almost solely inresearch centers, university hospitals and occasionally in highlyspecialized fertility centers.

An additional approach for semen measurements is described in U.S. Pat.Nos. 4,176,953 and 4,197,450, whose entire contents are incorporatedherein. These patents describe a method for measuring sperm motilityusing electro-optical means and an analog signal analyzer. A suspensionof sperm cells is continuously examined in a predetermined field inorder to detect variations in optical density by the motion of thesperm. An amplitude-modulated analog electrical signal is generated inresponse to the variations, and the peaks and valleys of this signal arecounted over a predetermined time period to provide an abstractparameter termed Sperm Motility Index (SMI). This parameter is relatedto motility and gives readings which are proportional to the number ofmotile cells multiplied by their respective velocity.

An automatic sperm analyzer called the Sperm Quality Analyzer (SQA),which provides the SMI parameter, has been on the market for a number ofyears. The analyzer is used in the following manner: a sperm specimen istaken up by a disposable chamber which has a rubber bulb at one end toaspirate the sample, and a thin measuring compartment at the other end.After aspirating the sample, the measuring compartment is inserted intothe SQA and the SMI of the sample is automatically determined. The SMIparameter, although useful in some applications, was not significantlyaccepted by the medical community as a viable alternative to theconventional microscopic semen measurements.

It is common knowledge that in some fields of veterinary fertilityanalysis, total sperm concentration (TSC), is evaluated by measuringoptical turbidity of the specimen. The physical principle behind thisapproach is that sperm cells are more opaque than the surroundingseminal plasma, and absorption of a light beam by the specimen istherefore proportional to the TSC.

For example, U.S. Pat. No. 4,632,562 discloses a method of measuringsperm density by measuring the optical absorbance of a sperm containingsample and relating the absorbance output signal to the density by usingat least three summing channels. The disclosed method is intended foruse in artificial insemination in the cattle breeding industry, andmeasures the optical absorbance in the range of 400-700 nm.

This technology however, has not and could not be adopted for human usefor the following reasons:

(1) Human sperm concentrations in the normal range (and even in higherthan normal cases), are more than an order of magnitude lower than inmost of their veterinary counterparts—where this technology has beenadopted.

(2) Human cases are treated even when sperm concentrations are far belowtheir normal levels. This of course is not the case for animals.Infertile animals are normally culled—in any case, they are not treatedfor infertility.

(3) TSC in humans is a parameter, which in itself, is totallyinsufficient for fertility investigations, and microscopic analysis isin any case required for all the other data in the standard semenanalysis protocol. To a large degree, this also holds for veterinaryapplications. This fact made optical absorption measurementssuperfluous, and no real effort has been invested in this field.

There is thus a need for a simple, objective technique for measuring TSCin human semen.

According to the WHO manual, sperm motility assessment (considered bymost to be the most important single semen parameter) can be carried outmanually using a grid system under the microscope or, alternatively, byuse of CASA.

CASA provides some advantages over manual methods. However, accuracy andprovision of quantitative data are totally dependent on precise semenpreparation techniques and instrument settings. These factors (highexpertise and sophisticated environment) along with the prohibitive costof such instrumentation, rule out for all practical purposes theirapplication for routine semen analysis.

U.S. Pat. No. 4,896,966 discloses a motility scanner for characterizingthe motion of sperm, bacteria and particles in fluid. The scannercomprises an optical system including a collimating lens, condensinglens, imaging lens and a pair of reflecting elements, a source ofillumination, radiation sensing means, signal processing means, anddisplay means. The imaging lens has a useful depth of field at itsobject plane of at least about 0.2 mm.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method formeasuring TSC.

It is a further object of the invention to provide a method fordetermining the motile sperm concentration (MSC) and % motility.

It is a still further object of the invention to provide a samplingdevice for use in the determination of semen parameters.

It is another object of the invention to provide a system for thedetermination of semen parameters.

In a first aspect of the invention, there is provided a method formeasuring the total sperm concentration (TSC) in a sample. The methodcomprises (i) placing the sample in a transparent container between asynchronically pulsed light source and a photodetector; and (ii)measuring the optical absorbance of the sample in the range of 800-1000mm, the TSC of the sample being proportional to the absorbance.

The method of the invention provides an objective measurement of TSCwhich is not dependent on image analysis, and which can measure humanTSC. However, the method may also be used to measure animal TSC.

In a second aspect of the invention, there is provided a sampling devicefor use in optically analyzing a biological fluid comprising:

-   -   (i) an aspirator for aspirating the fluid into the device;    -   (ii) a thin measuring chamber having an upper and lower wall,        the distance between the walls being in the range of 100-500        microns;    -   (iii) a thick measuring chamber having an upper and lower wall,        the distance between the walls being in the range of 0.5-3 cm;        and    -   (iv) means for excluding air from the measuring chambers.

In a preferred embodiment, the biological fluid is semen, mostpreferably human semen. The device serves both as a sampler and dualtest chamber, enabling simultaneous testing of TSC and MSC. No dilutionis required for any of the measurements. This not only saves labor butalso eliminates a significant source of errors—namely, dilutioninaccuracy.

The device also enables (when required) built-in visualizations of thespecimen without transferring it to a separate viewing chamber. Thethick chamber is also referred to as an optical densitometer.

In a third aspect of the invention, there is provided a method formeasuring motile sperm concentration (MSC) in a semen sample comprising:

-   -   (i) placing the sample in a transparent container between a        light source and a photodetector, wherein the sperm motion in        the sample modulates the light transmitted therethrough, thereby        generating a signal;    -   (ii) sampling the signal so as to produce a plurality of signal        samples;    -   (iii) selecting acceptable signals;    -   (iv) calculating an absolute value for each of the acceptable        signal samples;    -   (v) calculating an average a of the absolute values; and    -   (vi) calculating the MSC based on the average a.

It has now been discovered that analysis of waveforms of the analogsignals derived from a light beam which traverses a semen sample canprovide an indication of the MSC. Using appropriately selected criteria,excellent correlation was found to exist between the averaged areacovered by the waveform and the MSC. The MSC of a sperm sample isobtained in accordance with the invention by analyzing opticalproperties of the sample, which vary over time due to the motility ofthe sperm. This is in fact, the average signal amplitude in the relevantportions of the waveform, as will be described in more detail below.

In a fourth aspect of the invention, there is provided a method ofdetermining the average velocity (AV) of sperm cells comprising:

-   -   (i) obtaining a Sperm Motility Index (SMI) of the sperm cells as        defined in U.S. Pat. No. 4,176,953;    -   (ii) obtaining the MSC; and    -   (iii) calculating AV using an algebraic expression involving the        ratio SMI/MSC.

Reference is made here to U.S. Pat. No. 4,176,953 issued Dec. 4, 1979,and which has been implemented in various versions of Sperm QualityAnalyzers produced by Medical Electronic Systems, Israel. This patent,when applied to semen analysis, provides a parameter called SMI (SpermMotility Index). As disclosed in the above patent and proven in numeroussupporting studies, SMI is a function of both the concentration ofmotile cells (what is referred to as MSC) and their average velocity(AV). For the sake of simplification, we can say that SMI is a tofunction of MSCxAV, or AV is a function of SMI/MSC. The average velocityof a sperm sample can provide an indication of the quality of themotility of the sperm.

Not withstanding that which is stated above, SMI as a function of MSCand AV is more complex than a direct multiplication. After observing,analyzing and measuring over a hundred semen samples, the correctinter-relationship (formula) between them has been developed. In generalterms, the formula for extracting the average velocity can be definedas: AV=f(SMI/MSC), “f” being a polynomial of the third degree. Workingwith f(x)= 1/1000x³+ 1/10x²+0.89x, provided a correlation factor ofr=0.82.

It should be noted that most semen analysis protocols require data onthe % of sperm having progressive motility rather than their averagevelocity. Progressive motility is defined as those sperm having anaverage velocity of 5 microns/second or more. This parameter too, canreadily be extracted from the average velocity if a normal spread ofvelocities is assumed around the average. Even in cases where thevelocity spread is not normal, the error in calculating the % ofprogressively motile sperm is not significantly affected. Moreover, whendifferent minimal velocities are defined as progressively motile, thisvarying threshold is readily entered into the calculation, therebygiving extra flexibility in providing this parameter. This is importantwhen working in different diluting media, ambient temperatures or infact different species in vet measurements.

In a fifth aspect of the invention, there is provided a system foranalyzing sperm viability comprising:

-   -   (i) means for measuring TSC;    -   (ii) means for measuring MSC; and    -   (iii) a video visualization system.

The system of the invention combines the measurement of the two majorsperm parameters TSC and MSC, with the traditional visualization of thesperm, thus enabling acquiring the third parameter—sperm morphology. Ina preferred embodiment, TSC and/or MSC are determined according to themethods of the invention. In another preferred embodiment, the systemfurther comprises the sampling device of the invention.

It should be emphasized that there is a basic difference between thevideo visualization system used in the system of the invention and othersperm visualization systems (such as CASA). The other systems requireextremely high quality images because all their results are built onimage processing. In the present invention, on the other hand,visualization is used only as a complementary tool to view atypical orsuspect cases, to add confidence to processed results, to identifyspecific pathologies and to enable manual sperm morphology assessment,when required.

In order to fulfill these tasks, the video visualization system used inthe invention is designed as a compact, inexpensive subsystem, whichalthough of limited use as a stand-alone, precisely fills acomplementary role in the system of the invention. An additionalimportant advantage of the visualization system as compared tomicroscopic procedures, is that pipetting, preparation of slides,dilutions and filling of hemocytometers is unnecessary. Use, togetherwith the video visualization system, of the device of the invention,which doubles as a complete test chamber, obviates all of the above.These features, in effect permit and enable the use of the system of theinvention in any small clinic or even office environment.

The video visualization system allows one to obtain the followingsupplementary information regarding the tested sample:

1. Measurement of Very Low Sperm Concentrations

Measuring TSC at very low concentrations (below 5 million sperm/ml) isinherently limited in accuracy. This is due to the fact that lightabsorption by factors other than sperm cells, may become relativelysignificant at these low levels. Light absorption may be due to seminalplasma variability or to the presence of cells other than sperm. Thelatter include WBCs (white blood cells indicating infections) and otherimmature or non-spermic cells from various sources, etc. Since accordingto the invention TSC is measured by optical absorption, withoutvisualization there would be a possibility for ambiguity in the very lowranges due to the above mentioned considerations. When TSC is consideredimportant in the low ranges, visualization enables differentiationbetween the different cells contributing to the light absorption. SinceMSC is measured independently of light absorption, the % motility(MSC/TSC) can be calculated using the visually determined TSC parameter.

2. Identifying Foreign Cells in the Semen

The system is useful in identifying the presence of other cells whichmay have an effect on semen quality and/or assist in diagnosing patientailment. For instance, leukocytes indicate infection, immature cellsindicate a problem in spermatogenesis, agglutination may be due to anumber of causes, etc.

3. Manual Sperm Morphology Assessment

Although the system of the invention automatically assesses the % ofsperm with normal morphologies, it does so according to a given criteria(e.g. the WHO criteria). Regretfully this criteria is not universallyaccepted. Such universally accepted criteria do not yet exist, and areoften a factor of application. For example, morphology criteria for IVFand ICSI applications are normally far stricter than in normal cases.Other international standards (such as strict or Krueger criteria) arealso widely applied. Visualization allows the fertility practitioner toselect his own criteria as well as to identify the specific defectspresent (head deformity, tail problem, etc.)

4. Vasectomy Validation and Azoospermia Diagnosis

In order to fully validate the outcome of vasectomy or to obtain aconclusive diagnosis of azoospermia, it is necessary to determine thatthere are absolutely no sperm in the semen under evaluation. This isgenerally not possible with the light absorption technology, because theconcentrations that are to be measured can be very low. In this case,manual visualization is necessary in order to carefully scan largefields of view in search of individual sperm cells. The spermvisualization system used in the system of the invention is specificallytailored to optimally address these applications.

5. Hard Copy

The video visualization system enables “freezing” a given selected view(or a few views) which may then be printed and attached to the SemenAnalysis Report. This is of great value for consultations and validationof treatment efficacy. A by-product of the freezing option is viewingthe semen sample under static conditions. This strongly facilitatesanalysis and counting. In microscopic assessments, this can only be doneby demobilizing (killing) the sperm prior to viewing. Even then, alldead sperm will end up in one layer, a condition which normallycomplicates analysis due to high concentration and sperm overlap in thesaid layer.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, preferred embodiments will now be described, by way ofnon-limiting examples only, with reference to the accompanying drawings,in which:

FIG. 1 is block diagram illustrating one embodiment of the method ofmeasuring TSC according to the invention;

FIG. 2 is a perspective top view of one embodiment of a sampling deviceaccording to the invention;

FIG. 3 is a partial side sectional view of the device of FIG. 2;

FIG. 4 is a sectional view of the separating valve rotated 90° from theview of FIG. 3;

FIG. 5 is a schematic illustration of a system for semen analysisaccording to one embodiment of the invention;

FIG. 6 is a flow chart illustrating an algorithm for calculating theMSC;

FIG. 7 is a flow chart illustrating an algorithm for calculating theaverage velocity;

FIG. 8 illustrates a typical analog signal of motile sperm as a functionof time;

FIG. 9 is a correlation curve of the MSC with average analog signal; and

FIG. 10 is a block diagram illustrating one embodiment of a videovisualization system according to the invention.

DETAILED DESCRIPTION OF THE INVENTION Example 1

As stated above, the automatic optical measurement of TSC in human semensamples as opposed to animal samples has been hampered in the past dueto the low concentration of sperm cells. This, together with the highbackground electronic and optical noise due e.g. to seminal plasmavariability has prevented the application of methods routinely used inveterinary fertility analysis. The method of the present invention comesto overcome these obstacles by combining the following features:

-   -   (i) the sample is placed in a transparent container between a        synchronically pulsed light source and a synchronically enabled        photodetector. The use of a synchronically pulsed light source        and photodetector enables the distinction of sperm cells at low        concentrations over electronic noise levels.    -   (ii) measuring the optical absorbance of the sample in the range        of 800-1000 nm. It has been found that measuring the absorbance        in the near infrared region provides the optimal conditions for        obtaining strong absorption by sperm cells and low absorption by        seminal plasma. Preferably the measured range is 850-950 nm.        Most preferably, the range is 880-900 nm.

By using the method of the invention, the TSC of a sample may bedetermined as a function of the absorbance. Although the method of theinvention is preferably used with samples of human semen or human sperm,it may also be used with animal semen and animal sperm, preferably afterappropriate dilution.

An example of an optical system using one embodiment of the method ofthe invention is illustrated in FIG. 1. The system, indicated generallyby the numeral 2, comprises a light source 4, a photodetector 6 and asample holder 8 interposed therebetween. A preferred light source may bea fast-switching synchronically pulsed light emitting diode (LED) whichemits light in the near infrared region. The light source may becontrolled by a light intensity controller 10 which in turn is regulatedby a modulator 12. The photodetector is capable of detectingsynchronically pulsed light. The photodetector transmits the measuredanalog signals to a demodulator 14, which is also regulated by themodulator 12, and from there to output 16 of the signal in digital form.

The beam path through the sample is preferrably vertical. The length ofthe beam path through the sample is generally between 5 and 15 mm,preferably 10 mm. The sample holder must be fully transparent to lightwaves in the near infra-red region of between 800 and 1000 nm. Theplastic material from which the sample holder is made must be totallynon toxic to sperm cells. A preferred material is polystyrene PG-79. Thesample holder should preferably be designed to totally preventpenetration and forming of air bubbles in the sample, which interferewith the optical measurement.

By using the method of the invention, TSC detection levels down to appr.2 million cells/ml. have been achieved. This level already indicatesextreme semen pathology.

Example 2

FIG. 2 illustrates one embodiment of a sampling device 20 according tothe invention, for use in measuring semen. The device comprises ananterior optical viewing section 22, a posterior aspirating section 24and an intermediate air exclusion section 26.

The optical viewing section 22 comprises a thin measuring chamber 28 anda thick measuring chamber 30. The thin chamber is used to measure MSCand/or for visualization, while the thick chamber is used to measureTSC. In this way, multiple parameters can be measured simultaneouslyusing the same sampling device and sampling step.

The aspirating section 24 comprises a cylinder 32 and a plunger 34slidingly inserted therein. These parts match each other and function asin a standard syringe. This section serves for the aspiration of thesemen sample into the measuring chambers

The air exclusion section 26 comprises a separating valve 36 forseparation of the measuring chambers from the cylinder volume afterfilling. The aspirator, thin measuring chamber, thick measuring chamberand air exclusion section are all in fluid communication.

An adapter 38 in the form of a rectangular rail extends along one sideof the device 20 and serves for the correct sliding in and aligning ofthe device upon insertion into an optical instrument by which the sampleis evaluated. It also provides the mechanical support and stabilityrequired for precision electro-optical measurements.

The parts of the device may be seen more clearly in FIG. 3. The thinmeasuring chamber 28 is an internal cavity having an upper 40 and alower 42 parallel transparent wall through which the optical beam maypass. The distance between the walls is in the range of 100-500 microns,preferably 250-350 microns, most preferably approximately 300 microns.In the later case, the volume of liquid in the chamber is approximately25 μl. The anterior end 44 of the chamber has an aperture through whichthe sample may be drawn into the device. In the illustrated embodiment,the chamber is approximately 4 mm wide.

The thin measuring chamber serves for evaluation of sperm motility andmay be positioned between a light source e.g. opposite the lower wall 42and a photodetector e.g. opposite the upper wall 40. It will beunderstood that the light source and photodetector may also bepositioned on the opposite sides of the to chamber. A light beam istransmitted through the chamber containing a semen sample. The detectoron the other side of the chamber registers optical density variationscaused by moving sperm cells. The optical density variations aretranslated into an electrical signal by the photo-detector which is thenrouted to the electronic circuits to be filtered, digitized andprocessed so as to indicate the MSC. The thin measuring chamber may alsobe used with a video visualization system, as will be further explainedbelow.

The thick measuring chamber 30 has an upper 46 and a lower 48transparent wall through which an optical beam may pass. The distancebetween the walls is in the range of 0.5-3 cm, preferably 0.8-1.2 cm,most preferably approximately 1 cm. The approximate volume held by thethick compartment in the latter case would be approximately 0.5 ml.

This chamber serves for electro-optical absorption measurements of spermconcentration. A light beam, which may be the same or different fromthat of the thin chamber 28, is transmitted through the upper and lowerwalls of the chamber and detected by a photo-detector. The chambervolume should be completely filled with a sperm sample in order to avoidinaccuracies due to air bubbles. The attenuation of the light beam as itpasses through the chamber is proportional to the sperm concentration.The light beam intensity is measured after passing through the chamberand translated to units of TSC by electronic means. The order of thechambers in the sampling device may be exchanged.

The cylinder 32 is in fluid communication with the two measuringchambers 28 & 30, so that by drawing the plunger 34, fluid is drawn intothe chambers. This method of aspiration allows large sample volumes tobe aspirated into the device. In order to prevent air bubbles fromremaining in the measuring chambers, a separating valve 36 is interposedbetween the cylinder and the measuring chambers, and is in fluidcommunication with them. The valve is shown in detail in FIG. 4 andcomprises a piston 50 slidingly held in a valve housing 52. A connectingbore 54 connecting between the measuring chamber 30 and the cylinder 32passes through the piston 50.

When the valve is in the upper position, there is a connection betweenthe measuring chambers and the aspirating cylinder. Pressing the valvedown breaks that connection and ensures that no air remains in themeasuring chambers where the samples are measured and no leakage willoccur even when there is a temperature variation. This technique isequivalent to positive displacement since air is excluded from themeasured fluid volumes (except at the anterior end 44). This designenables working with samples of virtually all viscosities, while at thesame time preventing leakage and the penetration of air bubbles into thespecimen volumes to be analyzed.

Although the means for excluding air from the measuring chambers hasbeen exemplified by a separating valve, other means may also be used,such as a positive displacement pipette

All parts of the device may be manufactured from any material which isnot toxic to the measured cells. Preferably, the material is relativelycheap, such as plastic materials, so that the device can be disposable.An example of a polymer which may be used to produce the device ispolystyrene PG79. The separating valve, cylinder and piston may be madefrom polypropylene. The thin measuring compartment is by far the mosttoxi-sensitive part of the device due to the very high area to volumeratio of the seminal liquid in that section.

In order to aspirate a sample into the device 20, the tip 44 of the thinmeasuring compartment 28 is dipped approximately 5 mm deep into thesemen sample, which is then aspirated into the device past theseparating valve 36. Only app. 0.6 cc are required for a completefilling of the device. The separating valve is then pushed down, and thedevice may be inserted into an optical measuring apparatus.

Example 3

As mentioned above, determination of the MSC according to the inventionrequires the generation of a voltage signal which is proportional to theMSC. FIG. 5 shows one embodiment of a system for semen analysis capableof generating such a to signal.

An optical capillary 100 having a rectangular cross-section is used tohold a semen sample 102. The capillary 100 is illuminated with anincident light beam 105 produced by a light source 110. The capillary100 has an optical path of 300 μm through which the light beam 105passes. After passing through the capillary, the scattered beam 106 iscollimated by a round aperture 108 having a diameter of 70 μm. Thecollimated beam 107 impinges upon a photodetector 115. The photodetector115 produces an analog voltage signal 120 proportional to the intensityof the beam 107. The analog signal varies in time due to the motility ofthe sperm in the semen sample 102, as shown for example in FIG. 8. Theanalog signal 120 is inputted to an analog-to-digital converter 125 thatsamples the analog signal 120 at a rate of e.g. 8000 Hz and generates adigital output signal 128. The digital output signal may be stored in amemory 130. Sperm motion in the sample 102 leads to a modulation in theintensity of the beam 107, which in turn affects the analog signal 120and digital signal 128.

A processor 135 is configured to carry out an analysis of data stored inthe memory 130 in order to produce an analysis of the semen sample 102.The results of the analysis may be displayed on any display device suchas a CRT screen 140 of a personal computer 145, or on an internal LCDscreen 148 of the measuring device.

FIG. 6 shows a flow chart diagram for one embodiment of an algorithm forcalculating the MSC as carried out by e.g. the processor 135 of FIG. 5,in accordance with the invention.

In step 200, the digital signal 128 of FIG. 5 is digitally filtered inorder to remove high and low frequencies that are not relevant to thedominant frequency of the signal, which is determined by the motilitycharacteristics of the semen sample 102. This is done in order tooptimize the signal to noise ratio. The DC component of the signal 128is also removed. For human sperm samples, for example, the optimalrelevant frequency range was found to be between 5 and 30 Hz. In step205, digital samples having an absolute value below a firstpredetermined threshold, which may be determined empirically, areexcluded. In step 210 the same threshold value is subtracted from allremaining samples.

In step 215, a waveform selection procedure is carried out to discardall waveforms due to artifacts such as from non-relevant cells, etc. Apreferred embodiment of waveform selection with human sperm is toeliminate all waveforms not satisfying the following criteria:

Minimum height—10 millivolts.

Minimum width—37.5 milliseconds.

Maximum width—500 milliseconds.

Minimum rise/fall time—2.5 milliseconds.

The correct definition (and detection) of the beginning and end of spermassociated waveforms are defined as those where significant changes ofwaveform direction occur. The time difference between two such pointsdefines the time width of a given wave. The manner of selection may beunderstood by way of example with reference to FIG. 8 (not drawn toscale), which shows the amplitude of the analog signal (120 in FIG. 5)as a function of time. The threshold 302 is determined empirically toprovide optimal linearity between the output signal and themicroscopically measured MSC. The waveforms that are used for thecalculation of MSC are labeled 304, 305, 306 and 307. The otherwaveforms have been rejected for various reasons: 308 because its peakis less than the threshold; 310 because it is too wide; and 312 becauseit is too narrow.

In step 220 of FIG. 6 the absolute value of all selected samples iscalculated, and in step 225, the average a of the absolute values iscalculated. In step 230, the MSC of the sample 102 is calculated basedupon the average a. For example, it was found that the dependency of MSCon a can be described by a linear equation of the form:

MSC=αa

where α is an empirically derived constant. In a preferred embodiment,the to dependency of MSC on a may be described by a quadratic equationof the form:

MSC=Aa ² +Ba

With reference to FIG. 9, a specific human sperm sample was analyzed inaccordance with the invention. It was found that the dependency of MSCon a could be described by the following algebraic expression:

MSC=0.0047a ²+0.869a  (I)

A good linear correlation was found to exist for small values of a.Using formula (I), the correlation factor (r) for fresh sperm over theentire range was >0.98.

Analysis of treated semen samples with varying viscosity was alsoperformed using thawed samples, washed sperm, diluted samples (both in3% Sodium Citrate and Test Yolk buffer) as well as with samplescontaining up to 20% glycerol having artificially raised viscosity. Itwas found that varying sample viscosity (and therefore sperm velocity),did not significantly affect the correlation between MSC and averagesignal (“r” in all case remained above 0.96).

Using centrifugal enrichment techniques, a very wide range of motilehuman sperm concentrations were measured (up to 250 M/ml). Nosignificant saturation was found. The slight non-linearity at thehighest ranges is easily corrected by a simple second-degree polynomialcorrection—given above.

Analysis of bovine semen was also carried out and correlation factorsbetween bovine MSC and identically averaged signals (same methodology asfor humans) provided similarly excellent results. It is to be notedhowever, that bovine semen has to be diluted prior to measurements. Thisis due to their MSC being typically an order of magnitude above that ofhuman.

Example 4

As explained above, the average velocity is a function of SMI and MSC.

With reference to FIG. 7, the SMI is calculated in step 235. This may bedone, for example, as disclosed in U.S. Pat. No. 4,176,953, or using anSQA analyzer. In step 240 the MSC is calculated by any known method. Ina preferred embodiment, MSC is calculated by the algorithm of theinvention (see Example 3 above). In step 245 the average velocity AV iscalculated using an algebraic expression involving the ratio SMI/MSC. Inone embodiment AV is calculated using the algebraic expression:

${AV} = {{0.001\left( \frac{SMI}{MSC} \right)^{3}} + {0.1\left( \frac{SMI}{MSC} \right)^{2}} + {0.89\left( \frac{SMI}{MSC} \right)}}$

In step 250 the results are displayed on the display device 145 or 148(FIG. 5).

Example 5

One embodiment of a video visualization subsystem (VVS) which may beused with the analyzing system of the invention is illustrated in FIG.10. A semen sample 300 is placed before a diffused, phase contrastedilluminator 305. The sample may be held in a standard laboratory slideor smear, or may be held in a sampling device according to theinvention. Light from the illuminator 305 passes through the sample 300and through a switchable dual lens system 310, preferably withamplifications of 20 and 40. The amplified light is then conveyed to aminiature CCD video camera 315. The resulting image may be displayed ona built-in internal viewing screen 320 or on external displaying means325 such as PCs, screens, printing devices, etc.

In a preferred embodiment, the VVS is built around the sampling deviceof the invention, and particularly the thin measuring compartment. Theobject of this feature is that no extra preparations will be necessaryto incorporate this function to the normal testing procedure. One simplytakes the semen filled device on which the automated test is performedand inserts it—as such, into the viewing port. However, the VVS is notlimited to use with the sampling device of the invention, and may beused with standard laboratory slides or smears.

The front end of the VVS is similar to that of the microscope. Twoobjective lenses are selectable for optimizing magnification and fieldof view, according to the application (×20 or ×40). However, instead ofthe eyepieces of the microscope, the image from the objective isconveyed to a miniature CCD video camera. The size of the CCD (diagonal)is 6 mm. The viewing screen is a 100 mm LCD. This provides a videoamplification of app. 17. This in effect gives a potential overallamplification of 340 or 680. Although amplification factors of only 200and 400 are required, this set up is selected so that the aboveamplification could be reached in a much smaller construction. This isdesirable e.g. for a compact and robust desk-top unit (decreasing thespecified image distance decreases the amplification to what isrequired).

The lenses and their magnification set-up may be selected so that the“Working distance” (from object to lenses) can be varied to enablescanning throughout the whole depth of the thin measuring compartment(e.g. 300 microns). This is opposed to normal microscopic viewing whichdoes not require such scanning, because the object is normally enclosedin a slide which is just 20 microns deep and the whole depth can beviewed without scanning or refocusing.

As mentioned above, an overall amplification factor of 200 or 400 may beselected. An amplification of 400 will be the choice when it isnecessary to identify non-spermic cells (white blood cells, round cells,etc.), as well as to investigate and evaluate various morphologicalpathologies of sperm cells (agglutinations, immature cells, sperm heador tail defects, etc.). An amplification of 200 will be preferable forcell counting—irrespective of whether they are sperm or others. Thelower amplification provides a larger field of view (4 times larger) andthereby improved counting statistics. The possibility of freezing imagesgreatly enhances both applications.

In order to facilitate cell counts and acquire a truly quantitativeresult using the VVS, in a preferred embodiment a calibrated grid may becharted directly on the LCD viewing screen. The grid comprises 2 cmsquares which are equivalent to a pre-amplification size of 0.1 mm inthe semen filled measuring compartment (amplification factor of 200).This approach precludes the very difficult task of precisely charting aminute grid on the measuring compartment itself. The latter expensivesolution is incorporated in the Mackler Counting Chamber as well as someother hemacytometers—precluding their use as disposables. In the presentinvention this is unnecessary and the VVS allows the grid to be a partof the viewing screen.

The VVS may be useful in the following applications:

-   -   (a) Measuring very low sperm concentrations.    -   (b) Identifying foreign cells in the semen (other than sperm        cells).    -   (c) Manual morphology analysis according to any selected        criteria.    -   (d) Vasectomy efficacy validation.    -   (e) Diagnosing Azoospermia.    -   (f) On the spot comparison of computerized results with visual        analysis.    -   (g) Providing hard copy “Snap shots” of immobilized images of        various semen layers. The immobilization is achieved by        electronic freezing of the images.

1-29. (canceled)
 30. A method for measuring motile sperm concentration (MSC) in a semen sample, comprising: placing the sample in a transparent container between a light source and a photodetector, wherein the sperm motion in the sample modulates the light transmitted therethrough, thereby generating a signal; sampling the signal to produce a plurality of signal samples; selecting acceptable signals; calculating an absolute value for each of the acceptable signal samples; calculating an average a of the absolute values; and calculating the MSC based on the average a.
 31. The method according to claim 30, wherein the MSC is calculated according to the algebraic expression MSC=Aa²+Ba.
 32. The method according to claim 31, wherein the MSC is calculated according to the algebraic expression MSC=0.0047a²+0.869a. 